Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.
-
date post
21-Dec-2015 -
Category
Documents
-
view
214 -
download
0
Transcript of Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.
![Page 1: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/1.jpg)
Presented by: Andrew Nelson, R.J. Dealy,
and Nick Bishop
![Page 2: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/2.jpg)
Overview
• Intro• Lactococcus lactis
• Interleukins (cytokines)
• Quick summary of general process
• Relative Importance
• Materials and Methods• Outline of what was done
• Specific techniques used
• Results and Discussion• How and what results were
generated
• What was learned
![Page 3: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/3.jpg)
Lactococcus lactis
• Gram positive, cocci, 0.5-1.5μm• Used in the production of
fermented milk products (buttermilk, cheese, etc…)
• Noninvasive (can’t multiply in vivo), nonpathogenic
• Can serve as an antigen delivery vehicle
![Page 4: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/4.jpg)
Interleukins (cytokines)
• Cytokines (secreted signaling molecules)
• Immune system depends on them
• Can be useful immune response modulators for vaccines
• Administration of IL-2 and IL-6 has been shown to increase antibody responses to antigens
![Page 5: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/5.jpg)
What was done
• Constitutive expression strains were engineered to accumulate a test antigen (TTFC) and murine interleukins (cytokines) IL-2 and IL-6
• Mice were intranasally immunized with variations of these expression strains (as well as controls)
• Some recombinants were treated (killed) with mitomycin C prior to immunization
• Both systemic and mucosa anti-TTFC antibody responses were measured
![Page 6: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/6.jpg)
Relative Importance
• Treatment of disease• At this point, virtually all recombinant delivery systems had been
derived by infectious agents (Salmonella spp., Mycobacterium bovis, etc…)
• Cytokine delivery could enable tailored vaccines against particular pathogens.
![Page 7: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/7.jpg)
Materials and Methods
![Page 8: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/8.jpg)
Recombination of DNA
• PCR amplification using Vent polymerase
• DNA-modifying enzymes and restriction endonucleases used under standard conditions
• L. lactis transformed by electroporation of cells grown in presence of glycine
• Recombinant strains expressing TTFC, IL-2 and IL-6
![Page 9: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/9.jpg)
![Page 10: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/10.jpg)
Immunoblotting
• Cell walls were digested using a buffer solution and separated from the cells by centrifugation
• Proteins extracted using electrophoresis and electroblotted
• Transfer of TTFC and murine cytokines detected by immunoblotting (western blot)
![Page 11: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/11.jpg)
Preparation for Immunization• The bacterial strains which carried the
desired insert components were cultured, washed and resuspended prior to immunization
![Page 12: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/12.jpg)
Mitomycin PretreatmentPrior to immunization, cultured cells were treated with mitomycin C. After treatment, fewer than 1 in 104 cells remained viable.
![Page 13: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/13.jpg)
Immunization
• Groups of lady mice, 6-8 weeks old, were intranasally immunized with the modified L. lactis
Pretend this is a mouse.
![Page 14: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/14.jpg)
Detection of Antibodies• The presence of antibodies was detected using
enzyme-linked immunosorbent assay (ELISA). This method was developed using antibodies which react with serum antigens or antibodies and signal their presence
![Page 15: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/15.jpg)
This was done for both TTFC-specific antibodies and antilactococcal antibodies.
L. lactis
TTFC
![Page 16: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/16.jpg)
Assay of IgA
• Fecal pellets from the subjects were assyed for IgA concentration. IgA is the main antibody (immunoglobulin) in mucosal tissues
![Page 17: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/17.jpg)
Results• Feces of mice examined to determine if cytokines
would influence IgA in mucosal tissues• IgA levels in mice inoculated with different strains
of recombinant L. lactis showed the same results as the control groups
• Therefore, IgA levels in gastrointestinal tract were unaffected.
• But, blood serum antibody levels were affected….
![Page 18: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/18.jpg)
Mean anti-TTFC IgG levels
Increased levels in:
- TTFC + IL-2
- TTFC + IL-6
Recombinant bacterial strains from left to right:
TTFC, TTFC + IL-2, TTFC + IL-6, control non-expressor strain, control nonvaccinated group.
![Page 19: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/19.jpg)
Mean Anti-TTFC IgA Levels
Increased levels in:
- TTFC + IL-6
Recombinant bacterial strains from left to right:
TTFC, TTFC + IL-2, TTFC + IL-6, control non-expressor strain, control nonvaccinated group.
![Page 20: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/20.jpg)
Anti-TTFC antibody levels
a: IL-2
b: IL-6
-Inoculated mice with viable strains and mitomycin C killed strains of L. lactis
-TTFC antigen delivery does not require bacterial viability
- IL-2 and IL-6 cytokine delivery does require bacterial viability
![Page 21: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/21.jpg)
Results
So...
By demonstrating an increase in levels of IgA and IgG antibodies in mice inoculated with TTFC + IL-2 and TTFC + IL-6, it has been shown that…
The viable recombinant lactococci were successful in delivering cytokines to the immune system.
![Page 22: Presented by: Andrew Nelson, R.J. Dealy, and Nick Bishop.](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649d5e5503460f94a3d73f/html5/thumbnails/22.jpg)
Conclusions
• L. lactis can be used an effective method for delivering cytokines to the immune system via intranasal inoculation.
• This strategy may potentially be applied as a method of vaccination for a particular pathogen of interest.